Project Summary/Abstract Human pluripotent stem cells (hPSCs) retain the ability to generate any cell type in the human body, offering unprecedented access to a wide-array of human cell types. hPSCs are therefore a powerful model to study human neurodevelopmental diseases in vitro.57,59 Rett Syndrome (RTT), a devastating Autism-Spectrum- Disorder that affects 1 in 10,000 female births and causes severe regression by 18 months of age, is one such neurodevelopmental disorder.1,2 Hallmarks of RTT include an imbalance in inhibitory:excitatory synapses as well as excessive microglial pruning, but how these two pathologies are interconnected has yet to be fully explored. In this study, my goal is to decipher how inhibitory:excitatory synaptic imbalance in RTT is related to microglial-neuronal interplay using an iPSC-derived multi-cell type in vitro model containing both neurons and microglia. While there are established protocols for deriving neurons from hPSCs, to date there have been no developmentally-based differentiation schema for generating hPSC-derived microglia. Preliminary data shows that I can generate yolk sac primitive hematopoiesis in vitro to isolate microglial progenitors, and I have developed two different paradigms to generate microglial-like cells after co-culture with neurons. In this study, I aim to 1) compare the two paradigms to determine which generates microglial-like cells most similar to human primary microglia, and 2) use these cells to study the contribution of microglial-neuronal interaction in an iPSC-model of RTT. Specifically, I will investigate whether the inhibitory:excitatory synaptic imbalance in RTT is caused by a neuron-only phenotype or whether it is affected by microglial pruning. Through this study, I hope to generate bonafide human microglia through a novel, developmental paradigm, as well as answer a key mechanistic question behind RTT synaptic pathology.